Conformationally-preorganized, miniPEG-containing gamma-peptide nucleic acids

ABSTRACT

The present invention relates to γ-PNA monomers according to Formula I where substituent groups R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , B and P are defined as set forth in the specification. The invention also provides methodology for synthesizing compounds according to Formula I and methodology for synthesizing PNA oligomers that incorporate one or more Formula I monomers.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.14/921,755, filed Oct. 23, 2015 (now U.S. Pat. No. 10,093,700), which isa continuation of U.S. patent application Ser. No. 14/110,689, filedJan. 17, 2014 (now U.S. Pat. No. 9,193,758), which is a U.S. nationalstage of PCT/US2012/03259, filed Apr. 6, 2012, which claims priorityfrom U.S. provisional applications No. 61/516,812 and No. 61/516,838,both filed Apr. 8, 2011. The contents of these applications areincorporated herein by reference in their entirety.

GOVERNMENT LICENSE RIGHTS

This invention was made with government support under Grant No. GM76251,awarded by the National Institutes of Health, and Grant No. CHE-1012467,awarded by the National Service Foundation. The government has certainrights in this invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Dec. 11, 2015, isnamed 101700-0111_SL.txt and is 5,969 bytes in size.

BACKGROUND OF THE INVENTION

PNAs are a class of nucleic acid mimics in which the naturally occurringsugar phosphodiester backbone is replaced with N-(2-aminoethyl) glycineunits. See Nielsen, P. E.; et. al., Science 1991, 254, 1497-1500.Because of the homomorphous nature of the backbone and linker, PNAs canhybridize to complementary DNA and RNA through normal Watson-Crickbase-pairing just as the natural counterparts, but with higher affinityand sequence selectivity. See Egholm, M., et al., Nature 1993, 365,566-568.

PNAs are also capable of invading selected sequences of double-strandedDNA (dsDNA) attributed in large part to the lack of electrostaticrepulsion between the PNA and DNA strands. While the underlyingmechanism for high sequence selectivity of a PNA hybrid with either aDNA or RNA is not fully understood, structural studies suggested thathydration may play a key role in binding and selectivity. For instance,X-ray structural data of PNA-DNA and PNA-RNA duplexes indicates that amolecule of water bridges the amide proton in the backbone to theadjacent nucleobase rigidifying the PNAs backbone and preventingsequence mismatches thereby making the sequence mismatch lessaccommodating.

In addition the ability of PNAs to hybridize to DNA or RNA with highsequence selectivity, biochemical studies indicate that PNAs possesenhanced nucleolytic and proteolytic stability, most likely due to theirunnatural backbone that prevents or slows down the physiologicaldegradation of PNA's by proteases or nucleases.

Despite the many appealing features that make PNAs attractive asmolecular reagents for biology, biotechnology and medicine, PNAs havesome drawbacks as compared to other classes of oligonucleotides. PNAshave a charge neutral backbone as a result of which PNAs have poor watersolubility, the propensity to aggregate and adhere to surfaces andadhere to other macromolecules in a nonspecific manner. This inherentproperty of non-specific aggregation and surface adherence presents atechnical challenge for the handling and processing of PNAs.

While considerable efforts have been made to address these problems,several of the prior art efforts have focused on incorporating chargedamino acid residues at the termini or in the interior of a PNA oligomer,the inclusion of polar groups in the backbone, the replacement of theoriginal aminoethylglycyl backbone skeleton with a negatively-chargedscaffold, the conjugation of high molecular weight polyethylene glycol(PEG) to one of the oligomer termini, or fusion of a PNA to a DNA togenerate a chimeric oligomer to improve water solubility. However, thesechemical modifications are often achieved at the expense of bindingaffinity and/or sequence specificity.

Additionally, the high costs associated with synthesis of PNAs haslimited their incorporation as reagents routinely used in diagnosticassays, gene therapy and other biochemical assays.

SUMMARY OF THE INVENTION

The present invention addresses drawbacks of the conventional technologyby providing a hydrophilic PNA moiety with improved hybridizationproperties, water solubility and biocompatibility. More particularly,the invention relates to the design, synthesis, and uses of ahydrophilic (R)-miniPEG PNA unit having a polyethyleneglycol (miniPEG or“MP”) sidechain at the γ-carbon of the PNAs' backbone.

According to one embodiment, therefore, the invention provides compoundaccording to Formula I

In Formula I, B is a nucleic acid base selected from adenine, guanine,cytosine, thymine or uracil. Substituent groups R₁, R₂ and R₅ eachindependently are selected from the group consisting of H, linear orbranched (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl,(C₁-C₈)hydroxyalkyl, (C₃-C₈)aryl, (C₃-C₈)cycloalkyl,(C₃-C₈)aryl(C₁-C₆)alkylene, (C₃-C₈)cycloalkyl(C₁-C₆)alkylene,—CH₂—(OCH₂—CH₂)_(q)OP1, —CH₂—(OCH₂—CH₂)_(q)—NHP₁,—CH₂—(OCH₂—CH₂—O)_(q)—SP₁ and —CH₂—(SCH₂—CH₂)_(q)—SP₁.

Substituents R₃ and R₄ each independently are H while R₆ is selectedfrom the group consisting of H, linear or branched (C₁-C₈)alkyl,substituted or unsubstituted (C₃-C₈)aryl and (C₃-C₈)aryl(C₁-C₆)alkylene.

According to Formula I, P is selected from the group consisting of H,9-fluorenylmethyloxy carbonyl, Boc, benzyloxycarbonyl, tosylate, benzyl,alloc, trityl, dimethoxytrityl and monomethoxytrityl and substituent P₁is selected from the group consisting of H, (C₁-C₈)alkyl,(C₂-C₈)alkenyl, (C₂-C₈)alkynyl, (C₃-C₈)aryl, (C₃-C₈)cycloalkyl,(C₃-C₈)aryl(C₁-C₆)alkylene and (C₃-C₈)cycloalkyl(C₁-C₆)alkylene.Subscripts n and q are each independently integers between 0 and 10inclusive.

According to one embodiment, each of R₁ and R₂ in a Formula I compoundis independently —CH₂—O—(CH₂—CH₂—O)_(q)P₁. For some Formula I compoundseach of R₁ is —CH₂—(O—CH₂—CH₂—)_(n)OP₁ and R₂ is selected from the groupconsisting of H, linear or branched (C₁-C₈)alkyl, (C₂-C₈)alkenyl,(C₂-C₈)alkynyl, (C₁-C₈)hydroxyalkyl, (C₃-C₈)aryl, (C₃-C₈)cycloalkyl,(C₃-C₈)aryl(C₁-C₆)alkylene, (C₃-C₈)cycloalkyl(C₁-C₆)alkylene,—CH₂—(OCH₂—CH₂)_(q)—NHP1, —CH₂—(OCH₂—CH₂—O)_(q)—SP₁ and—CH₂—(SCH₂—CH₂)_(q)—SP₁. For certain Formula I compounds R₁ is—CH₂—(O—CH₂—CH₂—)_(q)OP₁, R₂ is H and substituent P₁ is H or(C₁-C₈)alkyl.

Formula I compounds are chiral. The stereochemical purity of a Formula Icompound is in the range from about 80% to about 99% at the Cγ-position.In one embodiment the stereochemical purity is at least 90% at theCγ-position. According to yet another embodiment the stereochemicalpurity of a Formula I compound is at least 99% at the Cγ-position.

The present invention also provides a method for preparing a compoundaccording to Formula I. According to the inventive method, a compound ofFormula II

(II) is contacted with a Formula III

(III) compound to obtain a compound according to Formula IV

The Formula IV compound is contacted with a compound according toFormula V

(V) to give a Formula I compound. Substituent groups B, R₁, R₂, R₃, R₄,R₅, R₆, P and P₁ are defined above. Substituent Y in Formula III isselected from the group consisting of bromine, iodine,4-toluenesulfonate and methanesulfonate.

According to the inventive synthetic methodology, the step of contactinga Formula IV compound with a Formula V compound is effected in thepresence of a coupling agent selected from the group consisting ofdicyclohexylcarbodiimide, carbonyldiimidazole,O-(benzotriazol-1-yl)-N,N,N′N′-tetramethyluronium hexafluorophosphate(HBTU), (benzotraizol-1-yloxy)tris(dimethylamino)phosphoniumhexafluorophosphate (BOP) andO-(7-azabenzotriazol-1-yl)-N,N,N′N′-tetramethyluroniumhexafluorophosphate (HATU) in a polar aprotic solvent.

In one embodiment, the present invention provides a method forsynthesizing a compound of Formula III by contacting

with a CH₃—(O—CH₂—CH₂—)_(q)OX group to obtain

The carboxylic acid group of the polyethyleneoxy product is furtherreduced to the corresponding alcohol; and then brought in contact with areagent to obtain the Formula III compound.

According to one embodiment the alcohol is brought in contact with areagent selected from the group consisting of methanesulfonyl chloride,4-toluenesulfonyl chloride and sodium iodide in an aprotic solvent. Whenthe alcohol is contacted with sodium iodide the contacting step iseffected in the presence of a catalyst, such as zirconium (IV) chloride.

In one embodiment the present invention provides a method forsynthesizing a peptide nucleic acid (PNA) oligomer having apre-determined sequence, by contacting a solid support with an allyllinker according to Formula VI

De-protecting the DMT group to obtain the corresponding alcohol which isthen brought in contact with a first PNA monomer or a γPNA monomerdepending on the PNA oligomer sequence. The carboxylic acid group of thefirst monomer is activated prior to contact with the allyl linker-resin.Following coupling of the first PNA residue to the resin deprotectingthe amino group of the first PNA residue.

Activating the carboxylic acid group of a second sequence specific PNAmonomer or γPNA monomer and contacting this activated carboxylic acidPNA with the amino group of the PNA residue attached to the resin. Thesteps described above are repeated to synthesize the peptide nucleicacid (PNA) oligomer comprising at least one γPNA monomer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A illustrates CD spectra of PNA5 and PNA2 (Inset) as a function oftemperature. Melting transition (T_(m)) of PNA2 through 5 as determinedby CD, monitored at 260 nm. The oligomer concentration was 5 μM,prepared in 10 mM sodium phosphate buffer at pH 7.4.

FIG. 1B is a graph correlating the stability of PNA oligomers to thenumber of inventive R-MP-γPNA monomers in the oligomer.

FIGS. 2A-2B illustrate UV-melting profiles of PNA-DNA (FIG. 2A) andPNA-RNA (FIG. 2B) hybrid duplexes at a strand concentration of 5 μM eachin 10 mM sodium phosphate buffer at pH 7.4. While both the heating andcooling runs were performed because they both have nearly identicalprofiles UV-melting for only the heating runs are shown.

FIG. 3 illustrates the correlation between Gibbs binding free energy(AG) and the number of miniPEG units in PNA-DNA and PNA-RNA duplexes.

FIG. 4 illustrates surface plamon resonance (SPR) sensorgrams (solidblack lines) and fits (dotted lines) for hybridization of PNA probes toimmobilized complementary DNA. Solutions contained 30 nM PNA. Error barsat t=420 sec illustrate standard deviations for three separate trials.

FIGS. 5A-5B show fluorescent spectra of PNA1X/PNA1Y (FIG. 5A) andPNA4X/PNA4Y (FIG. 5B) pairs at different concentrations. The sampleswere prepared by mixing equimolar ratios of the oligomers in 10 mMsodium phosphate buffer at pH 7.4. Samples were excited at 475 nm (FITCλ_(max)) and the emissions were recorded from 480 to 700 nm. The spectrawere normalized with respect to the FITC emission.

FIG. 6 illustrates the results of a non-denaturing gel-shift assay thatwas aimed at evaluating the extent of non-specific binding for anunmodified PNA oligomer (PNA6) and a oligomer containing the inventiveR-MP-γPNA monomer (PNA10). A drastic reduction in the intensity of theDNA band was observed with increasing concentrations of PNA6.

FIG. 7 illustrates a synthetic reaction scheme that does not requireprotection of nucleobases, wherein unprotected nucleobases are directlycoupled to a Boc- or a Fmoc-protected yPNA backbone.

FIG. 8 illustrates an efficient method for synthesizing theBoc-protected and/or Fmoc-protected PNA backbones, using Boc- andFmoc-protected amino acids, for example, Boc or Fmoc protected alanine,threonine, cysteine, or serine.

FIG. 9 illustrates a solid phase synthesis for Formula I PNA monomersusing Boc-protected (pathway (A)) and Fmoc-protected PNA (pathway (B))monomers.

FIGS. 10A and 10B illustrates the use of a novel allyl linker to connectthe first PNA building block to a solid resin support (FIG. 10A), andrelease of the final oligomer from the solid support under near neutralconditions by treating the resin with palladium tetrakistriphenylphosphine (Pd(PPh₃)₄) and a stoichiometric amount of morpholine(FIG. 10B).

FIG. 11 illustrates the synthesis of Boc-protected ^(R-MP)γPNA monomerscontaining all four natural nucleobases (A, C, G, T).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention concerns a new class ofconformationally-preorganized, MiniPEG-containing γPNA monomers thatpossess good water solubility, exhibit superior hybridizationproperties, biocompatibility, can readily invade double-stranded DNA andsecondary structures of RNA, and are capable of undergoing facilechemical diversification, such as by the introduction of functionallydiverse chemical groups at one or both termini of the PNA monomer orwithin the PNAs backbone. Thus, the invention provides compoundsaccording to Formula I, as well as methodology for synthesizing aFormula I γPNA monomer and also for synthesizing a PNA monomer havingone or more Formula I γPNA monomers.

Definitions

Within the context of the present invention, the term “miniPEG” or “MP”are used interchangeably and refer to a single poly-ethyleneglycol (PEG)unit or a polymer of PEG comprising from 2-50 PEG monomers. According toone embodiment, the term miniPEG includes without limitation a—CH₂—(OCH₂—CH₂)_(q)OP₁ group where subscript q is an integer between1-50 and P₁ is selected from the group consisting of H, (C₁-C₈)alkyl,(C₂-C₈)alkenyl, (C₂-C₈)alkynyl, (C₃-C₈)aryl, (C₃-C₈)cycloalkyl,(C₃-C₈)aryl(C₁-C₆)alkylene and (C₃-C₈)cycloalkyl(C₁-C₆)alkylene.Illustrative of miniPEG units include without limitation—CH₂—(OCH₂—CH₂)₁₋₄₅OH, —CH₂—(OCH₂—CH₂)₁₋₄₀OH, —CH₂—(OCH₂—CH₂)₁₋₃₅OH,—CH₂—(OCH₂—CH₂)₁₋₃₀OH, —CH₂—(OCH₂—CH₂)₁₋₂₅OH, —CH₂—(OCH₂—CH₂)₁₋₂₀OH,—CH₂—(OCH₂—CH₂)₁₋₁₅OH, —CH₂—(OCH₂—CH₂)₁₋₁₀OH, and —CH₂—(OCH₂—CH₂)₁₋₅OHgroups.

Further illustrative of the class minPEG are—CH₂—(OCH₂—CH₂)₁₋₄₅O(C₁-C₈)alkyl, —CH₂—(OCH₂—CH₂)₁₋₄₀(C₁-C₈)alkyl,—CH₂—(OCH₂—CH₂)₁₋₃₅O(C₁-C₈)alkyl, —CH₂—(OCH₂—CH₂)₁₋₃₀O(C₁-C₈)alkyl,—CH₂—(OCH₂—CH₂)₁₋₂₅O(C₁-C₈)alkyl, —CH₂—(OCH₂—CH₂)₁₋₂₀O(C₁-C₈)alkyl,—CH₂—(OCH₂—CH₂)₁₋₁₅O(C₁-C₈)alkyl, —CH₂—(OCH₂—CH₂)₁₋₁₀O(C₁-C₈)alkyl, and—CH₂—(OCH₂—CH₂)₁₋₅O(C₁-C₈)alkyl groups.

“Alkyl” refers to straight, branched chain, or cyclic hydrocarbyl groupsincluding from 1 to about 20 carbon atoms. For instance, an alkyl canhave from 1 to 10 carbon atoms, 1-8 carbon atoms, or 1 to 5 carbonatoms. Exemplary alkyl includes straight chain alkyl groups such asmethyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl,decyl, undecyl, dodecyl, and the like, and also includes branched chainisomers of straight chain alkyl groups, for example without limitation,—CH(CH₃)₂, —CH(CH₃)(CH₂CH₃), —CH(CH₂CH₃)₂, —C(CH₃)₃, —C(CH₂CH₃)₃,—CH₂CH(CH₃)₂, —CH₂CH(CH₃)(CH₂CH₃), —CH₂CH(CH₂CH₃)₂, —CH₂C(CH₃)₃,—CH₂C(CH₂CH₃)₃, —CH(CH₃)CH(CH₃)(CH₂CH₃), —CH₂CH₂CH(CH₃)₂,—CH₂CH₂CH(CH₃)(CH₂ CH₃), —CH₂CH₂CH(CH₂CH₃)₂, —CH₂CH₂C(CH₃)₃,—CH₂CH₂C(CH₂CH₃)₃, —CH(CH₃)CH₂CH(CH₃)₂, —CH(CH₃)CH(CH₃)CH(CH₃)₂, and thelike. Thus, alkyl groups include primary alkyl groups, secondary alkylgroups, and tertiary alkyl groups.

The phrase “substituted alkyl” refers to alkyl substituted at 1 or more,e.g., 1, 2, 3, 4, 5, or even 6 positions, which substituents areattached at any available atom to produce a stable compound, withsubstitution as described herein. “Optionally substituted alkyl” refersto alkyl or substituted alkyl.

Each of the terms “halogen,” “halide,” and “halo” refers to —F, —Cl,—Br, or —I.

The terms “alkylene” and “substituted alkylene” refer to divalent alkyland divalent substituted alkyl, respectively. Examples of alkyleneinclude without limitation, ethylene (—CH₂—CH₂—). “Optionallysubstituted alkylene” refers to alkylene or substituted alkylene.

“Alkene or alkenyl” refers to straight, branched chain, or cyclichydrocarbyl groups including from 2 to about 20 carbon atoms having oneor more carbon to carbon double bonds, such as 1 to 3, 1 to 2, or atleast one carbon to carbon double bond. “Substituted alkene” refers toalkene substituted at 1 or more, e.g., 1, 2, 3, 4, 5, or even 6positions, which substituents are attached at any available atom toproduce a stable compound, with substitution as described herein.“Optionally substituted alkene” refers to alkene or substituted alkene.

The term “alkenylene” refers to divalent alkene. Examples of alkenyleneinclude without limitation, ethenylene (—CH═CH—) and all stereoisomericand conformational isomeric forms thereof. “Substituted alkenylene”refers to divalent substituted alkene. “Optionally substitutedalkenylene” refers to alkenylene or substituted alkenylene.

“Alkyne or “alkynyl” refers to a straight or branched chain unsaturatedhydrocarbon having the indicated number of carbon atoms and at least onetriple bond. Examples of a (C₂-C₈)alkynyl group include, but are notlimited to, acetylene, propyne, 1-butyne, 2-butyne, 1-pentyne,2-pentyne, 1-hexyne, 2-hexyne, 3-hexyne, 1-heptyne, 2-heptyne,3-heptyne, 1-octyne, 2-octyne, 3-octyne and 4-octyne. An alkynyl groupcan be unsubstituted or optionally substituted with one or moresubstituents as described herein below.

The term “alkynylene” refers to divalent alkyne. Examples of alkynyleneinclude without limitation, ethynylene, propynylene. “Substitutedalkynylene” refers to divalent substituted alkyne.

The term “alkoxy” refers to an —O-alkyl group having the indicatednumber of carbon atoms. For example, a (C₁-C₆)alkoxy group includes—O-methyl (methoxy), —O-ethyl (ethoxy), —O-propyl (propoxy),—O-isopropyl (isopropoxy), —O-butyl (butoxy), —O-sec-butyl (sec-butoxy),—O-tert-butyl (tert-butoxy), —O-pentyl (pentoxy), —O-isopentyl(isopentoxy), —O— neopentyl (neopentoxy), —O-hexyl (hexyloxy),—O-isohexyl (isohexyloxy), and —O-neohexyl (neohexyloxy).

“Hydroxyalkyl” refers to a (C₁-C₁₀)alkyl group wherein one or more ofthe alkyl group's hydrogen atoms is replaced with an —OH group. Examplesof hydroxyalkyl groups include, but are not limited to, —CH₂OH,—CH₂CH₂OH, —CH₂CH₂CH₂OH, —CH₂CH₂CH₂CH₂OH, —CH₂CH₂CH₂CH₂CH₂OH,—CH₂CH₂CH₂CH₂CH₂CH₂OH, and branched versions thereof.

The term “ether” or “oxygen ether” refers to (C₁-C₁₀)alkyl group whereinone or more of the alkyl group's carbon atoms is replaced with an —O—group. The term ether includes —CH₂—(OCH₂—CH₂)_(q)OP1 compounds where P1is a protecting group, —H, or a (C₁-C₁₀)alkyl. Exemplary ethers includepolyethylene glycol, diethylether, methylhexyl ether and the like.

The term “thioether” refers to (C₁-C₁₀)alkyl group wherein one or moreof the alkyl group's carbon atoms is replaced with an —S— group. Theterm thioether includes —CH₂—(SCH₂—CH₂)_(q)—SP₁ compounds where P1 is aprotecting group, —H, or a (C₁-C₁₀)alkyl. Exemplary ethers includedimethylthioether, ethylmethyl thioether.

The term “aryl,” alone or in combination refers to an aromaticmonocyclic or bicyclic ring system such as phenyl or naphthyl. “Aryl”also includes aromatic ring systems that are optionally fused with acycloalkyl ring as herein defined.

A “substituted aryl” is an aryl that is independently substituted withone or more substituents attached at any available atom to produce astable compound, wherein the substituents are as described herein.“Optionally substituted aryl” refers to aryl or substituted aryl.

“Arylene” denotes divalent aryl, and “substituted arylene” refers todivalent substituted aryl. “Optionally substituted arylene” refers toarylene or substituted arylene.

The term “heteroatom” refers to N, O, and S. Inventive compounds thatcontain N or S atoms can be optionally oxidized to the correspondingN-oxide, sulfoxide or sulfone compounds.

The term “cycloalkyl” refer to monocyclic, bicyclic, tricyclic, orpolycyclic, 3- to 14-membered ring systems, which are either saturated,unsaturated or aromatic. The cycloalkyl group may be attached via anyatom. Cycloalkyl also contemplates fused rings wherein the cycloalkyl isfused to an aryl or hetroaryl ring as defined above. Representativeexamples of cycloalkyl include, but are not limited to cyclopropyl,cyclobutyl, cyclopentyl, and cyclohexyl. A cycloalkyl group can beunsubstituted or optionally substituted with one or more substituents asdescribed herein below.

The term “cycloalkylene” refers to divalent cycloalkyl. The term“optionally substituted cycloalkylene” refers to cycloalkylene that issubstituted with 1 to 3 substituents, e.g., 1, 2 or 3 substituents,attached at any available atom to produce a stable compound, wherein thesubstituents are as described herein.

The term ‘nitrile or cyano” can be used interchangeably and refer to a—CN group which is bound to a carbon atom of a heteroaryl ring, arylring and a heterocycloalkyl ring.

The term “oxo” refers to a═O atom attached to a saturated or unsaturated(C₃-C₈) cyclic or a (C₁-C₈) acyclic moiety. The ═O atom can be attachedto a carbon, sulfur, and nitrogen atom that is part of the cyclic oracyclic moiety.

The term “amine or amino” refers to an —NR^(d)R^(e) group wherein R^(d)and R^(e) each independently refer to a hydrogen, (C₁-C₈)alkyl, aryl,heteroaryl, heterocycloalkyl, (C₁-C₈)haloalkyl, and (C₁-C₆)hydroxyalkylgroup.

The term “amide” refers to a —NR′R″C(O)— group wherein R′ and R″ eachindependently refer to a hydrogen, (C₁-C₈)alkyl, or (C₃-C₆)aryl.

A “hydroxyl” or “hydroxy” refers to an —OH group.

The term “(C₃-C₈)aryl-(C₁-C₆)alkylene” refers to a divalent alkylenewherein one or more hydrogen atoms in the C₁-C₆ alkylene group isreplaced by a (C₃-C₈)aryl group. Examples of (C₃-C₈)aryl-(C₁-C₆)alkylenegroups include without limitation 1-phenylbutylene, phenyl-2-butylene,1-phenyl-2-methylpropylene, phenylmethylene, phenylpropylene, andnaphthylethylene.

The term “(C₃-C₈)cycloalkyl-(C₁-C₆)alkylene” refers to a divalentalkylene wherein one or more hydrogen atoms in the C₁-C₆ alkylene groupis replaced by a (C₃-C₈)cycloalkyl group. Examples of(C₃-C₈)cycloalkyl-(C₁-C₆)alkylene groups include without limitation1-cycloproylbutylene, cycloproyl-2-butylene,cyclopentyl-1-phenyl-2-methylpropylene, cyclobutylmethylene andcyclohexylpropylene.

A “peptide nucleic acid” refers to a DNA or RNA mimic in which the sugarphosphodiester backbone of the DNA or RNA is replaced by aN-(2-aminoethyl)glycine unit.

Some compounds described here can have asymmetric centers and thereforeexist in different enantiomeric and diastereomeric forms. A compound ofthe invention can be in the form of an optical isomer or a diastereomer.Accordingly, the invention encompasses compounds of the invention andtheir uses as described herein in the form of their optical isomers,diastereoisomers and mixtures thereof, including a racemic mixture.Optical isomers of the compounds of the invention can be obtained byknown techniques such as asymmetric synthesis, chiral chromatography,simulated moving bed technology or via chemical separation ofstereoisomers through the employment of optically active resolvingagents.

Unless otherwise indicated, “stereoisomer” means one stereoisomer of acompound that is substantially free of other stereoisomers of thatcompound. Thus, a stereomerically pure compound having one chiral centerwill be substantially free of the opposite enantiomer of the compound. Astereomerically pure compound having two chiral centers will besubstantially free of other diastereomers of the compound. A typicalstereomerically pure compound comprises greater than about 80% by weightof one stereoisomer of the compound and less than about 20% by weight ofother stereoisomers of the compound, for example greater than about 90%by weight of one stereoisomer of the compound and less than about 10% byweight of the other stereoisomers of the compound, or greater than about95% by weight of one stereoisomer of the compound and less than about 5%by weight of the other stereoisomers of the compound, or greater thanabout 97% by weight of one stereoisomer of the compound and less thanabout 3% by weight of the other stereoisomers of the compound.

If there is a discrepancy between a depicted structure and a name givento that structure, then the depicted structure controls. Additionally,if the stereochemistry of a structure or a portion of a structure is notindicated with, for example, bold or dashed lines, the structure orportion of the structure is to be interpreted as encompassing allstereoisomers of it. In some cases, however, where more than one chiralcenter exists, the structures and names may be represented as singleenantiomers to help describe the relative stereochemistry. Those skilledin the art of organic synthesis will know if the compounds are preparedas single enantiomers from the methods used to prepare them.

Compounds

The γ-PNA monomers of the present invention are conformationallypreorganized ethylene glycol containing compounds according to FormulaI.

For Formula I compounds, B is a nucleic acid base selected from adenine,guanine, cytosine, thymine or uracil. Each of groups R₁, R₂ and R₅ areindependently selected from the group consisting of H, linear orbranched (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, (C₃-C₈)aryl,(C₃-C₈)cycloalkyl, (C₃-C₈)aryl(C₁-C₆)alkylene,(C₃-C₈)cycloalkyl(C₁-C₆)alkylene, —CH₂—(OCH₂—CH₂)_(q)OP1,—CH₂—(OCH₂—CH₂)_(q)—NHP1, —CH₂—(OCH₂—CH₂)_(q)—SP₁ and—CH₂—(SCH₂—CH₂)_(q)—SP₁. According to one embodiment, R₁ and R₂ are eachindependently —CH₂—(OCH₂—CH₂)_(q)OP1. For instance, R₁ can be a—CH₂—(OCH₂—CH₂)_(q)OP1 group and R₂ can be selected from the groupconsisting of H, linear or branched (C₁-C₈)alkyl, (C₂-C₈)alkenyl,(C₂-C₈)alkynyl, (C₃-C₈)aryl, (C₃-C₈)cycloalkyl,(C₃-C₈)aryl(C₁-C₆)alkylene, (C₃-C₈)cycloalkyl(C₁-C₆)alkylene,—CH₂—(OCH₂—CH₂)_(q)—NHP1, —CH₂—(OCH₂—CH₂)_(q)—SP₁ and—CH₂—(SCH₂—CH₂)_(q)—SP₁. In one embodiment, R₁ is a—CH₂—(OCH₂—CH₂)_(q)OH group and subscript q is an integer between 1-25both integers inclusive, between 1-20 both integers inclusive, between1-15 both integers inclusive and between 1-10 both integers inclusive.

According to one embodiment, the present invention provides Formula Icompounds in which each of groups R₃ and R₄ independently is H. ForFormula I compounds R₆ is selected from the group consisting of H,linear or branched (C₁-C₈)alkyl, substituted or unsubstituted(C₃-C₈)aryl and (C₃-C₈)aryl(C₁-C₆)alkylene.

Substituent P on the terminal amino group of a Formula I compound can behydrogen or an amine protecting group. Exemplary of such protectinggroups include without limitation 9-fluorenylmethyloxy carbonyl (Fmoc),t-butyloxycarbonyl (Boc), benzhydryloxycarbonyl (Bhoc),benzyloxycarbonyl (Cbz), O-nitroveratryloxycarbonyl (Nvoc), benzyl (Bn),allyloxycarbonyl (alloc), trityl (Trt),1-(4,4-dimethyl-2,6-dioxacyclohexylidene)ethyl (Dde), diathiasuccinoyl(Dts), benzothiazole-2-sulfonyl (Bts), dimethoxytrityl (DMT) andmonomethoxytrityl (MMT) group.

For certain Formula I compounds substituent P₁ is selected from thegroup consisting of H, (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl,(C₃-C₈)aryl, (C₃-C₈)cycloalkyl, (C₃-C₈)aryl(C₁-C₆)alkylene and(C₃-C₈)cycloalkyl(C₁-C₆)alkylene. Subscripts n and q In Formula I areindependently integers between 0 and 50 both integers inclusive.According to one embodiment, subscript n is 1 while subscript p is aninteger between 1-45, preferably between 1-40, 1-35, 1-30, 1-25, 1-20,1-15, or 1-10.

According to one embodiment, the compound of formula I is as shown inChart 1:

CHART 1 Chemical compositions of the inventions.

MONOMERS P₁, P₂ P₃ X R₁, R₂, R₃, R₄ H Fmoc Boc Cbz Bn Tos Alloc Trt MMTDMT CH₃ Adenine Cytosine Guanine Thymine Modified nucleobasesFluorophores Aromatic ligands

 

Compounds conforming to Formula I are chiral by virtue of substituentgroup diversity at C-γ. A typical stereomerically pure Formula Icompound comprises greater than about 80% by weight of one stereoisomerof the compound and less than about 20% by weight of other stereoisomersof the compound. According to an embodiment, a stereomerically pureFormula I compound comprises greater than about 90% by weight of onestereoisomer of the compound and less than about 10% by weight of theother stereoisomers of the compound, or greater than about 95% by weightof one stereoisomer of the compound and less than about 5% by weight ofthe other stereoisomers of the compound, or greater than about 97% byweight of one stereoisomer of the compound and less than about 3% byweight of the other stereoisomers of the compound or greater than orequal to about 99% by weight of one stereoisomer of the compound andless than or equal to about 1% by weight of the other stereoisomerrespectively.

While MP and larger molecular-weight polyethylene glycol (PEG) unitshave been incorporated into a number of macromolecular systemsincluding, for example, peptides and proteins, nucleic acids,carbohydrates, synthetic polymers, dendrimers, liposomes, andnanoparticles, the present inventors unexpectedly found that theintroduction of a diethylene glycol group, commonly referred to as‘miniPEG’ or MP, in the backbone of PNA enhanced the aqueous solubility,biocompatibility and binding specificity along with reduction inaggregation and nonspecific binding of the PNA.

The PNA backbone offers a choice of three sites (C-α, C-β and C-γ), forintroducing a miniPEG (MP) group. Previous studies by the presentinventors have indicated that installation of a chiral center atposition C-γ within the PNA backbone induces helical organization(helicity) in the oligomer and provides a means for fine-tuning thethermodynamic stability of PNAs. The helical conformation adopted by anoligomer containing PNA monomers depends in part on the stereochemistryof the PNA monomers used. Two helical conformations are possible,namely, a right-handed conformation and a left-handed conformation.γPNAs prepared from L-amino acids adopt a right-handed helix, whilethose prepared from D-amino acids adopt a left-handed helix. However,bioanalytical studies indicate that only the right-handed helical γPNAshybridize to DNA and RNA with high affinity and sequence selectivity.

Synthesis

A. General Synthetic Protocols

Traditional routes for synthesizing PNAs have been tedious, involvingthe preparation of protected nucleobases—A, C, and G, and the use oftoxic chemicals and multiple steps to obtain an orthogonally protectedPNA monomer that can be used for synthesizing oligomers using a resin.As illustrated in FIG. 7, the present inventors have developed syntheticmethodologies that do not require protection of nucleobases. Rather, PNAmonomers according to Formula I are readily prepared using cheap,commercially available, unprotected nucleobases that are directlycoupled to a Boc- or a Fmoc-protected γPNA backbone.

Also provided is an efficient method for synthesizing the Boc-protectedand/or Fmoc-protected PNA backbones. Compared to the traditionalMitsunobu synthetic route used in the preparation of PNA backbones,synthesis of PNA backbones according to the methods described herein isaccomplished in a few simple steps from commercially available andrelatively cheap Boc- and Fmoc-protected amino acids, for example, Bocor Fmoc protected alanine, threonine, cysteine, or serine according tothe protocol (FIG. 8). Following this method, no elaborate columnchromatography purification is necessary to obtain PNA backbones thathave the required purity for coupling to unprotected nucleobases.

The present invention also provides an optimized solid-phase reactionsequence for synthesizing PNA containing oligomers that is moreefficient and reduces or eliminates a number of hazardous chemicaltransformation steps that routinely accompany traditional solid phasesynthesis (FIG. 9). Synthetic methodologies described herein, have ledto significant cost-reductions in the production of γPNA monomers andoligomers.

The synthetic protocol illustrated in FIG. 9 is optimized to efficientlycouple PNA monomers according to Formula I to a solid resin support withminimal side-reactions (less than 1%) or cross-coupling reactionsbetween the unprotected, exocyclic amino groups of adenine, cytosine, orguanine nucleobase and the activated carboxyl group of a PNA monomer.Because solid phase synthesis according to the inventive protocol usesunprotected nucleobases no deprotection of the nucleobases in the finaloligomer product are necessary prior to cleavage of the oligomer fromthe solid support. Additionally, pyridine neutralization and cappingsteps necessary for solid phase synthesis of DNA or RNA oligomers usingconventional methods are omitted in the present method with no effect onthe overall yield or purity of the final MP-γPNA oligomers.

Bypassing these steps not only significantly reduces the synthesis time,but also reduces the costs of oligomer synthesis and costs associatedwith disposal of hazardous wastes, such as pyridine and aceticanhydride, omitted from the protocol shown in FIG. 9.

FIG. 9 illustrates a solid phase synthesis for Formula I PNA monomersusing Boc-protected ((A) Boc SPPS Chemistry) and Fmoc-protected PNAmonomers ((B) Fmoc SPPS Chemistry). As stated above, one advantage ofcarrying out oligomer synthesis using a solid support is that it permitsin situ neutralization of the ammonium ion generated by trifluoroaceticacid (TFA) cleavage of the Boc protecting group. According to thepresent inventors in situ neutralization is superior to the standard,pyridine wash/neutralization sequence used conventionally because itimproves the overall yield and purity of MP-γPNA oligomers.

Another advantage of the synthetic method according to the presentinvention is the use of a C-terminal thioester activated PNA monomer incoupling reactions. Traditional synthetic routes do not employC-terminal thioester monomers for synthesis because of the ensuingintramolecular esterification and N-terminal truncation. In contrast,oligomer synthesis using a method according to the present inventiondoes not suffer from these drawbacks. This is so because neutralizationof the ammonium ion is carried out in situ and also due the greaterrigidity of the γ-modified PNA oligomers than their achiralcounterparts. Enhanced oligomer rigidity disfavors intramolecularesterification and N-terminal truncation products.

As stated above, the use of PNA monomers that have unprotectednucleobases during solid phase synthesis of an oligomer permits cleavageof the oligomer product from the resin support under mild conditions.According to one embodiment, the inventors have developed a novel allyllinker to connect the first PNA building block to the solid resinsupport. See FIG. 10A. The main advantage of the allyl linker is that itpermits the release of the final oligomer from the solid support undernear neutral conditions by treating the resin with palladium tetrakistriphenylphosphine (Pd(PPh₃)₄) and stoichiometric amount of morpholine(FIG. 10B).

PNA oligomers are important molecular tools in analytical assays and astherapeutic and diagnostic reagents for the treatment and detection ofgenetic diseases. Many diagnostic assays rely on sequence specifichybridization of the PNA oligomer to single stranded or duplex DNA orRNA. Other assays use a chemical probe covalently attached to the PNAoligomer to detect a biological macromolecule of interest. Both assaymethods rely on the subsequent release of the PNA-DNA or PNA-RNA hybrid,or the release of the probe-biological macromolecule complex from thesolid surface to permit their detection and quantitation.

Reagents traditionally used to release the PNA complexes from the solidsupport, however, are harsh and unsuitable for use with many biologicalsamples. The present inventors have addressed this problem by developinga novel allyl linker to attach PNA oligomers to a solid support so as tofacilitate the gentle release of the PNA-biocomplex formed during theassay under near neutral conditions.

B. Synthesis of Specific γPNA Monomers

In one embodiment, Boc-protected^(R-MP)γPNA monomers containing all fournatural nucleobases (A, C, G, T) were synthesized according to theprocedures outlined in FIG. 11.

As illustrated in FIG. 11, alkylation of Boc-protected L-serine (1) with1-bromo-2-(2-methoxyethoxy)ethane or 2-(2-methoxyethoxy)ethane methanesulfonate (2) was carried out as follows. To a vigorously stirred,chilled solution of DMF containing 2 equivalents of sodium hydride wasslowly added compound (1), followed by addition of1-bromo-2-(2-methoxyethoxy)ethane or or 2-(2-methoxyethoxy)ethanemethane sulfonate (2). After stirring at 0° C. for 1 hr, the mixture wasquenched by addition of water at 0° C. The solvents (DMF and water) wereremoved under reduced pressure at room temperature. Water was added tothe crude mixture and the pH was adjusted to ˜3 using 5% HCl. Theaqueous solution was extracted with ethyl acetate and dried over Na₂SO₄.The resultant product is pegylated Boc-protected serine, compound 3which is obtained with high optical purity.

Both the stoichiometry and order of addition of reagents were determinedto be important for obtaining an optically pure product. Slow additionof Boc-serine is necessary to ensure complete deprotonation of thecarboxyl group prior to removal of the hydroxyl proton. Formation of thecarboxylate anion reduces the acidity of the α-proton making it lesssusceptible to deprotonation by base.

Esterification of the alkylated product (3) followed by reduction withsodium borohydride yields the corresponding alcohol, serinol (4). Theconversion of the carboxylic acid moiety to an alcohol renders theCa-proton inert to deprotonation and racemization in subsequent reactionsteps. The serinol (4) was allowed to react with sodium iodide in thepresence of zirconium (IV) chloride (ZrCl₄) as a catalyst to obtain thecorresponding iodide (5). Subsequent displacement of the iodide by ethylglycinate yielded the PNA backbone (6).

Dicyclohexylcarbodiimide (DCC) mediated coupling of 6 with theappropriate carboxymethylnucleobases (A, C, G, and T), followed byhydrolysis of the resulting ester group gave the desired Formula I γPNAmonomers (8a-d).

The optical purities of key intermediates and final γPNA monomersaccording to Formula I were determined by ¹⁹F-NMR following chemicalderivatization as described in the literature. See Seco et al., Chem.Rev. 2004, 104, 17-117. Gas chromatography coupled to mass spectrometricdetection (GC/MS) has been described in the literature to determine theenantiomeric excess (ee) of chiral α-PNA monomers and their oligomers.See Corradini et al., Tetrahedron: Asymmetry 1999, 10, 2063-2066.

The present inventors found ¹⁹F-NMR to be a convenient and accuratealternative method for determining the ee values for Formula I γPNAmonomers and synthetic intermediates of γPNA monomers. Analysis by¹⁹F-NMR required removal of the Boc-protecting group and subsequentcoupling of the free amine group of a Formula I γPNA monomer a syntheticintermediate of γPNA monomer with(+)-1-methoxy-1-(trifluoromethyl)phenylacetyl chloride (MTPA-Cl,Mosher's reagent).

Boc-D-serine was used as the starting reagent to synthesize thecorresponding PNA stereoisomer (^(S-MP)γPNA monomer), which is requiredas a control to quantify the enantiomeric excess of the desired of^(R-MP)γPNA monomer. Inspection of the ¹⁹F-NMR spectral trace for MTPAderivatized ^(R-MP)γPNA monomer and ^(S-MP)γPNA monomer revealed notraces of the ^(S-MP)γPNA monomer indicating that the desired Formula Icompound is optically pure. Based on the spectral data it was concludedthat the desired Formula I PNA monomer had an optical purity of 99% ee,within the detection limit of ¹⁹F-NMR.

While thymine ^(R-MP)γPNA monomer showed two peaks for rotamers at−68.80 and −68.95 ppm in the NMR spectrum, the corresponding thymine^(S-MP)γPNA showed only one rotamer. The existence of the two rotamersfor thymine ^(R-MP)γPNA monomer is unclear.

γPNA monomers manufactured according to synthetic protocols describedabove have enantiomeric purity of at least 90% by weight of onestereoisomer of the compound and less than about 10% by weight of theother stereoisomer of the compound, or greater than about 95% by weightof one stereoisomer of the compound and less than about 5% by weight ofthe other stereoisomer of the compound, or greater than about 97% byweight of one stereoisomer of the compound and less than about 3% byweight of the other stereoisomer of the compound or greater than orequal to about 99% by weight of one stereoisomer of the compound andless than or equal to about 1% by weight of the other stereoisomerrespectively.

^(R-MP)γPNA monomers based on the L-alanine scaffold were synthesized asdescribed by Rapireddy et al., J. Am. Chem. Soc. 2007, 129, 15596-15600and He et al., J. Am. Chem. Soc. 2009, 131, 12088-12090.

While L-alanine-derived γPNA (^(S-Ala)γPNA) oligomers are able to invademixed-sequence double helical B-form DNA (B-DNA) and are promising asantisense and antigene reagents, the ^(S-Ala)γPNAs are poorly soluble inwater and have a tendency to aggregate, presumably due to thecharge-neutral backbone and hydrophobic character of the γ-Me. Accordingto one embodiment, therefore, the replacement of the side chain methylgroup with miniPEG, for example, an ethylene glycol unit [(OCH₂CH₂)_(n),where n=1-10] at C-γ results in a (R)miniPEG PNA monomer according toFormula I. Introducing the (R)miniPEG PNA monomer into a oligomer chaininduces a right-handed helix in the resultant PNA oligomer. Sucholigomers have improved water solubility and reduced aggregation whileretaining superior hybridization properties.

Biochemical Analysis

To evaluate whether a PNA oligomer containing one or more γPNA monomersaccording to Formula I influence the conformation and hybridizationproperties of PNA oligomer or influence the water solubility andaggregation properties of a PNA oligomer, the present inventorssynthesized PNA oligomers whose sequences are shown in Table 1 below.

TABLE 1 Sequence of PNA oligomers #MP SEQ ID Oligomer Sequence units NO:PNA1 H-GCATGTTTGA-NH₂  0  1 PNA2 H-GCATGTTTGA-NH₂  1  2 PNA3H-GCATGTTTGA-NH₂  3  3 PNA4 H-GCATGTTTGA-NH₂  5  4 PNA5 H-GCATGTTTGA-NH₂10  5 PNA6 H-ACGGGTAGAATAACAT-NH₂  0  6 PNA7 H-ACGGGTAGAATAACAT-NH₂  1 7 PNA8 H-ACGGGTAGAATAACAT-NH₂  3  8 PNA9 H-ACGGGTAGAATAACAT-NH₂  5  9PNA10 H-ACGGGTAGAATAACAT-NH₂  8 10 PNA1XH-^(L)Orn(X)-^(L)Lys-GCATGTTTGA-NH₂  0  1 PNA1YH-^(L)Lys-GCATGTTTGA-^(L)Orn(Y)-NH₂  0  1 PNA4XH-^(L)Orn(X)-^(L)Lys-GCATGTTTGA-NH₂  5  4 PNA4YH-^(L)Lys-GCATGTTTGA-^(L)Orn(Y)-NH₂  5  4 Underlined letter indicatesR-MP-γ-backbone modification. X = fluorescein (FITC), Y =tetramethylrhodamine (TAMRA).

The first set of oligomers (PNA1 through 5), were designed to test theeffects of miniPEG on the conformation and hybridization properties ofPNA. The second set of oligomers (PNA6 through 10), was designed to testthe effect of miniPEG on water solubility. A hexadecameric sequence ischosen for the aqueous solubility study because such a sequencerepresents a statistical length that would be required to target aunique site within the mammalian genome or transcriptome. The third setincluded two oligomers (PNA1 and 4). Each oligomer in this set wasdesigned to test the effect of miniPEG on self-aggregation tendency ofPNA containing oligomers using Förster Resonance Energy Transfer (FRET).Thus, PAN's 1 and 4 were separately linked to fluorescein (FITC) at theN-terminus (PNA1X and PNA4X) and tetramethylrhodamine (TAMRA) group atthe C-terminus (PNA1Y and PNA4) of each oligomer. A lysine residue wasintroduced at the C-terminus to improves water-solubility and aid in thepurification and characterization of the labeled oligomerss.

All PNA oligomers, those with and without MP side-chains, aresynthesized on solid-support according to the protocols described hereinor published in the literature. Unlike PNA's with modifications made atthe α-backbone that require further optimization of the solid phaseresin reaction coupling conditions in order to minimize racemization, noprecautions or modification of the synthetic protocol are necessary forcoupling of the inventive Formula I^(R-MP)γPNA monomers on the resin.

Moreover, after coupling the last monomer the resultant oligomer can bereadily cleaved from the resin and precipitated with ethyl ether. Theair-dried pellets of the crude oligomers are dissolved inwater/acetonitrile mixture (80/20), and purified by reverse-phase HPLCand characterized by MALDI-TOF mass spectrometry.

1. Effect of MiniPEG of Oligomer Conformation & Hybridization

PNA1 through 5 oligomers were analyzed by CD spectroscopy to determinethe effect of minPEG on the conformation of PNA oligomers. Consistentwith the earlier findings (Dragulescu-Andrasi, A. et al.; J. Am. Chem.Soc. 2006, 128, 10258-10267), no CD signals were observed within thenucleobase absorption regions for PNA1 that does not contain a Formula IR-MP-γPNA group. See FIG. 1A. This observation indicates that this PNAoligomer either (i) does not adopt a helical conformation, or (ii) hasan equal proportion of a right-handed and left-handed helix in theanalytical sample.

However, PNA2 through 5 show distinct exciton coupling patterns in theCD spectrum with two distinct minima's at 242 and 280 nm and twomaxima's at 220 and 260 nm. The observed CD pattern is characteristic ofa right-handed helix. See FIG. 3B. The addition of miniPEG units did notalter the amplitude of the CD signals. However, the addition of miniPEGdoes alter the wavelengths of maxima and minima, shifting it towardsthat of the PNA-DNA and PNA-RNA double helices (FIG. 3B).

Moreover, a gradual dip at the 242 nm minimum generally indicates atightening in the helical pitch of the oligomer from one that resemblesthat of a PNA-PNA duplex with 18 base-pairs per turn to one thatresembles that of a PNA-DNA duplex with 13 base-pairs per turn. Overall,the CD profiles of PNA2 through 5 are similar to those of thecorresponding PNA-DNA and PNA-RNA hybrid duplexes (FIG. 3B), the majordifference in the CD trace being the amplitude which is roughly doubledfor the duplex as compared to individual PNA strand.

Without ascribing to a particular theory, this doubling of amplitude islikely due to the higher concentration of bases in the hybrid duplex(approximately twice the concentration), than that of the individual PNAstrand. Taken together, these results show that a single, (R)-MP unitinstalled at the γ-backbone is sufficient to preorganize PNA into aright-handed helix.

While incorporation of additional miniPEG units does not further improvebase-stacking, as is apparent from the similarities in the CDamplitudes, the presence of additional miniPEG's does help to tightenthe helical pitch of the oligomers making them more rigid and compact.This is apparent from the temperature-dependent CD measurements, whichshowed a less dramatic reduction in the signal amplitude as a functionof temperature change for the PNAS oligomer consisting of ten R-MP-γPNAgroups as compared to the PNA2 oligomer having a single R-MP-γPNA group(FIG. 4). Even at a temperature as high as 80° C., a distinct CD profileis obtained for PNAS, indicating that base-stacking is occurring forthis oligomer at a temperature of 80° C. In contrast, PNA2 is completelydenatured at this temperature.

Thus, the overall stability of the oligomers increases linearly with thenumber of MP units incorporated (FIG. 1B). The fact that PNAS adopts ahelical conformation most closely resembling that of a PNA-DNA or aPNA-RNA duplex suggests that it can hybridize to DNA and RNA moreeffectively than the other oligomers in this series.

2. Effect of MiniPEG on Thermal Stability of Oligomers

UV-melting experiments were performed to determine the effect of MP onthe thermal stability of PNA oligomers following hybridization to DNA oran RNA. FIG. 3A illustrates that the incorporation of a single miniPEGside-chain stabilized a PNA-DNA duplex by 4° C. The extent of thermalstabilization gradually increased with additional minPEG units. However,increase in thermal stability tapers off to a value of about 2.3° C. perunit for the fully-modified oligomer, that is an oligomer made up ofR-MP-γPNA groups only (e.g., PNA5).

A similar pattern is observed for a R-MP-γPNA-RNA duplex, but theobserved increase in thermal stability is lower for a R-MP-γPNA-RNAduplex as compared to a R-MP-γPNA-DNA duplex (FIG. 3B). The enhancementin thermal stability of a R-MP-γPNA-RNA duplex is only 3° C. for thefirst R-MP-γPNA-monomer that is incorporated into the PNA oligomer andthis gain in thermal stability reduces to about 1.2° C. per R-MP-γPNAmonomer for an oligomer made entirely R-MP-γPNA (PNA5). In contrast thegain in thermal stability is about 2.3° C./unit for R-MP-γPNA-DNAduplexes.

It was further observed that while unmodified PNA1 binds more tightly toRNA than to DNA (differential T_(m) (ΔT_(m)) of 10° C.), thefully-modified miniPEG PNA5 displayed identical thermal stability withboth RNA as well as with DNA. The apparent lack for preferential bindingshown by PNA5 is not clearly understood but it may be due to rigidity ofthe PNA5 oligomer's backbone.

Without being bound to a particular theory, the present inventorsbelieve that because PNA5 is more rigid and tightly wound when comparedto PNA1 the rigid backbone limits conformational freedom necessary toaccommodate the DNA and/or RNA template strands. Under suchcircumstances, the DNA and RNA strands taking part in hybridizationthemselves are forced to undergo a conformational change necessary toaccommodate the ^(R-MP)γPNA helix. The above hypothesis provides anexplanation why an ^(S-Ala)γPNA-DNA prefers a P-form helix, a helicalstructure that is intermediate between the A- and B-form DNA. It is alsoclear that the hybridization of a ^(R-MP)γPNA to DNA and RNA requiresthe DNA and RNA moieties to conformationally alter to accommodate γPNAexigencies rather than the other way around.

Because the RNA strand is less accommodating to conformational changes,its hybridization to a fully modified ^(R-MPγ)PNA oligomer is lessfacile than hybridization of a DNA to the fully modified ^(R-MPγ)PNAoligomer.

Further insights related to the contribution of miniPEG to the stabilityof the PNA-DNA duplex was obtained from van't Hoff analysis. Data inTable 2 show the thermodynamic parameters associated with hybridizationof PNA1 through 5 to a complementary DNA or RNA strand.

TABLE 2 Thermodynamic parameters for PNA-DNA and PNA-RNA duplexesPNA-DNA^(†) PNA-RNA^(‡) −ΔH° −TΔS° −ΔG° −ΔH° −TΔS° −ΔG° Oligo (kJ/mol)(kJ/mol) (kJ/mol) K_(d) (kJ/mol) (kJ/mol) (kJ/mol) K_(d) PNA1 273 ± 5 224 ± 5  49 ± 1* 2.5 × 10⁻⁹  289 229 60 3.5 × 10⁻¹¹ PNA2 319 ± 18 263 ±16 54 ± 1  3.2 × 10⁻¹⁰ 333 232 68 1.2 × 10⁻¹² PNA3 316 ± 11 256 ± 11 59± 1* 5.1 × 10⁻¹¹ 350 280 71 4.3 × 10⁻¹³ PNA4 329 ± 14 265 ± 12 65 ± 1 3.5 × 10⁻¹² 356 283 73 1.7 × 10⁻¹³ PNA5 372 ± 11 294 ± 10 78 ± 2  4.6 ×10⁻¹⁴ 365 287 78 2.1 × 10⁻¹⁴ ^(†)The averages of three trials (2 fromconcentration-dependence measurements + 1 from UV-melting curvefitting). ^(‡)UV-melting curve fitting. *Standard deviation is less than1 kJ/mol. Temperature = 298K.

The results show that the Gibbs binding free energy (ΔG°) increasesapproximately linearly with increase in the number of miniPEG units forPNA-DNA duplexes, while increase in ΔG° is sigmoidal for PNA-RNAduplexes (FIGS. 3A and 3B).

The incorporation of a single miniPEG unit results in a net gain inbinding free energy of about 5 kJ/mol for the PNA-DNA duplex and is lessthan 5 KJ/mol for a PNA-RNA duplex. The gain in binding free energy,moreover, is not linearly correlated to the number of ^(R-MP)γPNAmonomers in the PNA oligomer. Rather, most of the net gain in bindingfree energy is from the first two ^(R-MP)γPNA monomers and decreases asmore ^(R-MP)γPNA monomers are introduced in the PNA oligomer of thePNA-RNA duplex. Additionally, a reduction in the equilibriumdissociation constant (K_(d)) by nearly five orders of magnitude wasobserved for a PNA5-DNA duplex while a decrease of about three orders ofmagnitude is observed for PNA5-RNA as compared to the PNA1-DNA andPNA1-RNA duplexes.

The binding free energy gain is believed to predominantly be fromenthalpic contributions for both PNA-DNA and PNA-RNA duplexes as isshown by the gradual increase in the ΔH° term with the number of minPEGunits present in the PNA. Further support that the gain in binding freeenergy is predominantly from enthalpic contributions stems from theobservation that single-stranded PNA's adopt a compact globular form,presumably to minimize exposure of the hydrophobic core of nucleobasesand the charge-neutral backbone to the aqueous solvent. It follows,therefore, that an enthalpic penalty would be incurred for unfolding thecollapsed (globular) PNA in order to adopt the helical structure neededto participate in hybridization to a complementary DNA or RNA. Removalof this penalty by inducing a helical structure through the use of theminiPEG modified γ-PNA according to Formula I would translate to a morefavorable enthalpic change during hybridization. See Table 2.

According to the present inventors, an additional enthalpic benefit ofthe modified backbone may be arise due to the formation of a network ofstructured water molecules that bridge the backbone amide protons to theadjacent nucleobases, stabilizing interactions that are more pertinentin a γPNA-DNA duplex than in a traditional PNA-DNA or PNA-PNA duplexes.

Surface plasmon resonance (SPR) analysis is used to study thehybridization kinetics of ^(R-MP)γPNA-DNA and ^(R-MP)γPNA-RNA duplexes.Briefly, SPR was performed as follows. According to one embodiment, thePNA probe was immobilized to the chip while the DNA target was capturedfrom solution. In another embodiment, a biotinylated version of the DNAtarget is immobilized on a streptavidin-conjugated, carboxymethylateddextran chip at a relatively low surface density (ca. 100 responseunits) of DNA targets to limit mass transport effects on the associationkinetics. Solutions containing 10-50 nM PNA oligomers are allowed toflow over the chip for about 420 seconds, at which point the flow isswitched to a PNA-free buffer to allow net dissociation of thehybridized PNA.

Individual sensorgrams for the unmodified (PNA1) and ^(R-MP)γ-modified(PNA2 through 5) oligomers at 30 nM concentration are shown in FIG. 6.While small variations are observed in the association kinetics, singlymodified PNA2 appears to bind approximately twice as fast as theunmodified PNA. Fitting the data to a 1:1 binding model yieldsassociation rate constants (k_(a)) that range from 4.7×10⁵M⁻¹s⁻¹ to9.7×10⁵M⁻¹s⁻¹ (Table 3).

Table 3. The association rate constant (k_(a)), dissociation rateconstant (k_(d)), and equilibrium dissociation constant (K_(d)) forhybridization of PNA probes with a complementary DNA target.

Oligomer k_(a) (M⁻¹s⁻¹) k_(d) (s⁻¹) K_(d) (M) PNA1 4.7 × 10⁵ 13.0 × 10⁻⁴2.8 × 10⁻⁹ PNA2 9.7 × 10⁵  4.1 × 10⁻⁴ 4.2 × 10⁻¹⁰ PNA3 6.2 × 10⁵  1.9 ×10⁻⁴ 3.0 × 10⁻¹⁰ PNA4 6.6 × 10⁵  0.3 × 10^(−4 †) 4.1 × 10^(−11 †) PNA58.0 × 10⁵  0.4 × 10^(−4 †) 5.4 × 10^(−11 †) † Indicates uncertainty dueto the calculated value approaching the limits of detection of theinstrument.

In contrast, significantly greater variability was seen in thedissociation phase of the experiment, with the dissociation rateconstant (k_(d)) varying by at least a factor of 50. Equilibriumdissociation constants (K_(d)) calculated from the ratio of thedissociation and association rate constants are also given in Table 3.Unmodified PNA1 and fully-modified PNAS have K_(d)=2.8 nM and 54 pM,respectively. The K_(d) values for PNA1-3 determined by SPR (Table 3)are similar to those determined by UV melting experiments (Table 2).However, increasing divergences are observed for PNA4 and PNAS, with theSPR-derived values being 12- and 1200-fold greater, respectively thanthe K_(d) values determined by UV melting experiments (Table 2).

This differences are attributed to the very small degrees ofdissociation observed within the timescale of the SPR experiment.However these small differences in the degree of dissociation introducea large uncertainty during the dissociation of the duplex and give riseto the differences in K_(d) values.

In the above example, SPR results clearly demonstrate that enhancedaffinity of the ^(R) ^(_) ^(MP)γPNAs are due to the significantly slowerdissociation kinetics of PNA oligomers containing one or more of the^(R) ^(_) ^(MP)γPNA monomers. Thus, the helical preorganization of themodified PNA may have a smaller contribution to faster hybridizationkinetics than previously proposed. That is, hybridization is likely torequire some structural reorganization of the complementary DNA strand,negating to some extent the benefit of pre-organizing the PNA oligomerto helical form.

CD, NMR, and X-ray data have shown that γPNAs derived from L-amino acidsadopt a right-handed helix, and that the helix becomes more rigid asmore γ-chiral units are added in the backbone. One would thereforeexpect a fully-modified PNA5 to hybridize to DNA and RNA targets withgreater sequence selectivity than PNA1. To verify this hypothesis,thermal stabilities of PNA5-DNA and PNA5-RNA duplexes containingperfectly-matched (PM) and single-base mismatched (MM) targets weredetermined and compared to those from an earlier study with PNA1-DNA andPNA1-RNA duplexes. The results show that despite the strong bindingaffinity, PNA5 is able to discriminate between closely relatedsequences. The ΔTm ranges from −17 to −21° C. for PNA5-DNA and −16 to−20° C. for PNA5-RNA containing a single-base mismatch (X═C, G, T), ascompared to −10 to −14° C. for PNA1-DNA and −11 to −18° C. for PNA1-RNAduplex (Table 4). The level of sequence discrimination is greater forPNA5-DNA than for PNA1-DNA, and similar, if not slightly better, forPNA5-RNA as compared to PNA1-RNA. This result is consistent with PNA5adopting a more rigid helical motif, which is less accommodating tostructural mismatches as compared to PNA1.

TABLE 4 Sequence mismatch discriminationPNA1: H-GCATGTTTGA-^(L)Lys-NH₂ (SEQ ID NO: 1)PNA5: H-GCATGTTTGA-^(L)Lys-NH₂ (SEQ ID NO: 5) DNA: 3′-CGTACAXACT-5, X =A, C, G, T (SEQ ID NO: 11) RNA: 3′-CGUACAXACU-5, X = A, C, G, U (SEQID NO: 12) T_(m) (° C.) T_(m) (° C.) X-T PNA1-DNA* PNA5-DNA PNA1-RNA*PNA5-RNA A-T 45 68 55 68 C<>T 31 (−14)^(†) 47 (−21) 37 (−18) 48 (−20)G<>T 31 (−14) 48 (−20) 44 (−11) 52 (−16) T(U)<>T 35 (−10) 51 (−17)40 (−15) 48 (−20) *The data for PNA1-DNA and PNA1-RNA mismatched bindingwas taken from Dragulescu-Andrasi, A.; et al., J. Am. Chem. Soc. 2006,128, 10258-10267. ^(†)The value in the parenthesis indicates ΔT_(m)between the perfect match and mismatch.

Effect of MiniPEG on Aqueous Solubility and Aggregation

To determine whether inclusion of miniPEG in the backbone of a Formula IPNA has an effect on water solubility of the resultant oligomer,saturating concentrations of PNA6 through 10 (Table 1) were prepared inwater and the concentrations of each solution was measured byUV-spectroscopy. The incorporation of a single MP unit enhances thesolubility of PNA6 by nearly 2-fold (Table 5). The solubility of theoligomers is further improved, albeit to a smaller extent, withadditional MP units.

TABLE 5 Saturated concentrations of PNA oligomers #MP Sat. conc.Oligomer units (mM) PNA6 0 39 PNA7 1 76 PNA8 3 108 PNA9 5 350 PNA10 8>500

FRET was used to study whether incorporation of a miniPEG unit in thebackbone of PNA can help reduce aggregation. Different concentrations ofunmodified PNA1X/PNA1Y and homologous γ-modified PNA4X/PNA4Y pairs(Table 1) are prepared by mixing equimolar ratios of the individualoligomers in sodium phosphate buffer. The samples were excited at 475nm, the λ_(max) of FITC, and emission was recorded from 480 to 700 nm.Upon aggregation, in which the oligomers bearing FITC and TAMRA comeinto contact with one another, excitation at 475 nm leads to energytransfer from FITC to TAMRA because of the proximity of the twochromophores. Comparison of the FRET efficiencies of the two systems atdifferent concentrations, therefore, can provide an assessment of theeffect of miniPEG on intermolecular interaction of PNA's.

As illustrated by FIG. 5A, when the concentration for each unmodifiedPNA oligomer is as low as 1 μM, a small but noticeable emission appearedat 580 nm, indicating some aggregation between PNA1X and PNA1Y. Theextent of aggregation is further intensified with increasingconcentrations of oligomers, apparent from the fluorescent intensity ofTAMRA at ˜580 nm upon excitation of the FITC donor at 475 nm.

In contrast, at a concentration of 20 μM, the point at which nearly 70%FRET efficiency is observed for unmodified PNA1X/PNA1Y pair, about 5%FRET efficiency is observed for the γ-modified PNA4X/PNA4Y pair. Theseresults indicate that the γ-modified PNA pair does not interact witheach other as much as the unmodified PNAs. The distinction is alsoapparent from photographs of the samples illuminated using ashort-wavelength (254 nm), hand-held UV-lamp. The PNA1X/PNA1Y solutiondisplayed a light orange emission at room temperature and yellow-greenhue at 90° C., an indication of the aggregate dissociating upon heating.In contrast, the PNA4X/PNA4Y solution displayed the same color,yellow-green, at room temperature as well as at 90° C., indicating thatthe oligomers are well dispersed even at room temperature. Thus, theR-MP-γ-modification imparts not only enhanced solubility to PNA, butalso suppresses aggregation.

It has been documented that at moderate concentrations, PNA tends toaggregate and stick to surfaces and other macromolecules in anonspecific manner. Such interactions can lead to off-target binding andcytotoxic effects, when employed in the cellular context. Among themacromolecules that PNA is known to interact nonspecifically with arenucleic acids and proteins.

To assess the extent of off-target binding of PNA and ^(R-MP)γPNA, agel-shift assay is performed. In this case, a DNA fragment, 171 bp inlength, is incubated with different concentrations of PNA6 and PNA10(Table 1) in 10 mM sodium phosphate buffer at 37° C. for 16 hr. The twooligomers contain identical nucleobase sequence but differ from anotherat the γ-backbone. PNA6 is unmodified, whereas PNA10 is modified atevery other position with R-MP-γ side-chain. Following incubation, thesamples are separated on non-denaturing polyacrylamide gel and stainedwith SYBR-Gold.

Since the target does not contain a complementary sequence to theoligomers, no binding is expected to take place, in which case theintensity of the DNA band should remain fairly constant, independent ofthe PNA6 and PNA10 concentrations. Instead, a drastic reduction in theintensity of the DNA band is observed with increasing concentrations ofPNA6 (FIG. 6). At 10 μM (corresponding to a PNA/DNA ratio of 25:1) orhigher, the DNA band completely disappeared from the gel.

In contrast, for γ-modified PNA10 the intensity of the DNA bandsremained fairly constant even at a concentration as high as 20 μM(PNA/DNA ratio of 50:1). This result is consistent with the solubilityand FRET data, indicating that incorporation of miniPEG at theγ-backbone not only improves the hybridization properties and watersolubility of PNA but also helps to reduce nonspecific binding withother macromolecules as well.

Gamma-backbone modified PNA's according to Formula I as well asoligomers containing the Formula I PNA's are provided, in accordancewith the invention, to improve design and utility of PNA-basedtherapeutic and diagnostics. For instance, improvements in hybridizationproperties can enable R-MPγPNAs to invade double helical DNA andstructured RNA that may not be permissible with other oligonucleotidemimics. Enhancements in water solubility will facilitate the handlingand processing of PNA while lessening the concerns for nonspecificbinding and cytotoxic effects. Improvements in these areas, along withthe flexibility of synthesis whereby other chemical functionalities canbe installed at the γ-backbone with ease, will expand the utility of PNAinto other scientific disciplines, including drug discovery andnanotechnology.

What is claimed is:
 1. A compound according to Formula VIII

wherein, P is selected from the group consisting of hydrogen (H),9-fluorenylmethyloxycarbonyl (Fmoc), t-butyloxycarbonyl (Boc),benzyloxycarbonyl (Cbz), benzyl (Bn), tosylate (Tos), allyloxycarbonyl(alloc), benzhydryloxycarbonyl (Bhoc), O-nitroveratryloxycarbonyl(Nvoc), 1-(4,4-dimethyl-2,6-dioxacyclohexylidene)ethyl (Dde),diathiasuccinoyl (Dts), benzothiazole-2-sulfonyl (Bts), trityl (Trt),monomethoxytrityl (MMT), and dimethoxytrityl (DMT); each of R₁ and R₂ isindependently selected from the group consisting of hydrogen (H), linearor branched (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl,(C₁-C₈)hydroxylalkyl, (C₃-C₈)aryl, (C₃-C₈)cycloalkyl,(C₃-C₈)aryl(C₁-C₆)alkylene, (C₃-C₈)cycloalkyl(C₁-C₆)alkylene,

provided that at least one of R₁ and R₂ is selected from the groupconsisting of

each of R₃ and R₄ is independently selected from the group consisting ofhydrogen (H),

R₅ is selected from the group consisting of hydrogen (H), linear orbranched (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl,(C₁-C₈)hydroxylalkyl, (C₃-C₈)aryl, (C₃-C₈)cycloalkyl,(C₃-C₈)aryl(C₁-C₆)alkylene, and (C₃-C₈)cycloalkyl(C₁-C₆)alkylene; R₇ isselected from the group consisting of hydrogen (H), linear or branched(C₁-C₈)alkyl, substituted or unsubstituted (C₃-C₈)aryl, and(C₁-C₆)alkylene; P₂ is selected from the group consisting of hydrogen(H), linear or branched (C₁-C₈)alkyl, 9-fluorenylmethyloxycarbonyl(Fmoc), t-butyloxycarbonyl (Boc), benzyloxycarbonyl (Cbz), benzyl (Bn),tosylate (Tos), allyloxycarbonyl (alloc), trityl (Trt),monomethoxytrityl (MMT), and dimethoxytrityl (DMT); P₃ is selected fromthe group consisting of hydrogen (H), linear or branched (C₁-C₈)alkyl,(C₂-C₈)alkenyl, (C₂-C₈)alkynyl, (C₃-C₈)aryl, (C₃-C₈)cycloalkyl,(C₃-C₈)aryl(C₁-C₆)alkylene, and (C₃-C₈)cycloalkyl(C₁-C₆)alkylene; x isan integer from 1 to 10, inclusive; and y is an integer from 1 to 9,inclusive.
 2. The compound of claim 1, wherein R₁ is—CH₂—O[(CH₂)₂—O—]_(X)R₈; each of R₂, R₃ and R₄ is hydrogen (H); whereinx is an integer from 1 to 4 inclusive; R₅ is selected from the groupconsisting of hydrogen (H) and methyl; R₇ is selected from the groupconsisting of hydrogen (H), methyl, ethyl, and t-butyl; and R₈ isselected from the group consisting of hydrogen (H), methyl, ethyl, andt-butyl.
 3. The compound of claim 2, wherein R₅ is hydrogen (H); R₇ ismethyl, ethyl or t-butyl; R₈ is methyl, ethyl, or t-butyl and P is9-fluorenylmethyloxycarbonyl (Fmoc) or t-butyloxycarbonyl (Boc).
 4. Thecompound of claim 3 having the formula VIIIa-1:

wherein, x is 1, 2 or 3; R₇ is methyl, ethyl or t-butyl, and R₈ ismethyl, ethyl or t-butyl.
 5. The compound of claim 4 having the formulaVIIIa-2 or VIIIa-3:

wherein R₇ is methyl, ethyl or t-butyl.
 6. The compound of claim 5,having an optical purity of at least 99% enantomeric excess (ee).
 7. Thecompound of claim 3 having the formula VIIIa-1T:

wherein, x is 1, 2 or 3; R₇ is methyl or ethyl and R₈ is methyl orethyl.
 8. The compound of claim 7 having the formula VIIIa-2T orVIIIa-3T:

wherein R₇ is methyl or ethyl.
 9. The compound of claim 8, having anoptical purity of at least 99% enantomeric excess (ee).
 10. The compoundof claim 1, wherein R₂ is —CH₂—O[(CH₂)₂—O—]_(X)R₈; each of R₁, R₃ and R₄is H, wherein x is an integer from 1 to 4 inclusive; R₅ is selected fromthe group consisting of hydrogen (H) and methyl; R₇ is selected from thegroup consisting of hydrogen (H), methyl, ethyl, and t-butyl; and R₈ isselected from the group consisting of hydrogen (H), methyl, ethyl, andt-butyl.
 11. The compound of claim 10, wherein R₅ is hydrogen (H); R₇ ismethyl, ethyl or t-butyl; R₈ is methyl, ethyl or t-butyl, and P is9-fluorenylmethyloxycarbonyl (Fmoc) or t-butyloxycarbonyl (Boc).
 12. Thecompound of claim 11, having the formula VIIIa-4:

wherein, x is 1, 2 or 3; R₇ is methyl, ethyl or t-butyl, and R₈ isselected from methyl, ethyl or t-butyl.
 13. The compound of claim 12,having the formula VIIIa-5 or VIIIa-6:

wherein R₇ is methyl, ethyl or t-butyl.
 14. The compound of claim 13,having an optical purity of at least 99% enantomeric excess (ee). 15.The compound of claim 11 having the formula VIIIa-4T:

wherein, x is 1, 2 or 3; R₇ is methyl or ethyl and R₈ is selected frommethyl or ethyl.
 16. The compound of claim 15 having the formulaVIIIa-5T or VIIIa-6T:

wherein R₇ is methyl or ethyl.
 17. The compound of claim 16, having anoptical purity of at least 99% enantomeric excess (ee).